Summary Cadherins are expressed in virtually all solid tissues and play a key role in a wide range of physiological and pathological processes. These calcium-dependent transmembrane molecules cluster at sites of cell-cell contacts where they mediate cell adhesion and signaling, which subsequently influence motility, differentiation, and carcinogenesis. When a cell-cell contact is formed, cadherins expressed on neighboring cells interact through their extracellular domain while their cytoplasmic domain interacts with the cytoskeleton through the catenin family of armadillo cadherin- binding proteins. The central hypothesis of this application is that cadherin/cadherin interactions on adjoining cells are regulated by the binding of specific proteins, such as 1-catenin, to the cytoplasmic domain of cadherins. This hypothesis finds support in previous structural data suggesting that integrins - cell-matrix adhesion molecules - switch from a low-affinity to a high-affinity conformation following binding of cytoplasmic proteins. We are in a unique position to test this hypothesis of enhanced homotypic cadherin affinity mediated by binding of cytoplasmic proteins in living cells because we have available 2 key tools developed in our complementary laboratories: (I) cell lines harboring human E-cadherin mutants derived from patients with hereditary diffuse gastric cancer (HDGC). Our recent results show that these disease-causing mutations prevent binding of specific proteins to their cytoplasmic domain and correlate with weakened global cell-cell adhesion and (II) an all-live cell single-molecule force spectroscopy assay that can probe cadherin/cadherin interactions between two adjoining cells at single-molecule resolution. The proposed research is high-risk because - while predicted for a long time - a direct demonstration of enhanced receptor-ligand affinity mediated by cytoplasmic proteins at the level of a single transmembrane molecule and in live cells has not been shown previously, and in particular, has not been presented for cadherins. By achieving the goals here proposed, we will contribute to fill the gap toward the understating of the mechanisms linking E-cadherin deregulation to cancer initiation and progression. PUBLIC HEALTH RELEVANCE: E-cadherin is critical for the maintenance of tissue architecture and its loss is associated with invasion and metastasis during cancer progression. The study of early invasive cancers in carriers of E-cadherin germline mutations demonstrates that its deregulation is also an initiating event in tumorigenesis. The mortality rate associated to epithelial cancers strongly increases when tumor cells are able to invade through the epithelial basal membrane. E-cadherin loss plays a pivotal role in this process and is considered a clinical turning point in carcinoma progression and metastization. Because of that, studies aimed at elucidating E-cadherin function and regulation have become of critical relevance in oncobiology research. Experimental evidence supports the idea that deregulation of cell adhesion is a necessary condition to promote cell invasion, so that by unraveling the molecular mechanisms that govern E-cadherin-dependent cell adhesion new molecular targets for invasive cancers could be identified.